Glucagon is synthesized in the pancreas. It is a highly conserved polypeptide consisting of a single chain of 29 amino acids, with a molecular weight of 3485 Da. Recombinant glucagon is expressed in E. coli and purified to at least 98% pure prior to use. Proteolytic removal of the amino-terminal histidine residue leads to loss of the biological activity. Glucagon has a helical conformation in the crystalline state, while in dilute aqueous solutions it has a random coil conformation with 15% alpha helix at the C-terminal end.
Pharmacologically, glucagon increases the concentration of glucose in the blood. The first six amino acids at the N-terminus of the glucagon molecule bind to specific receptors on liver cells. This leads to an increase in the production of cAMP, which facilitates the catabolism of stored glycogen and increases hepatic gluconeogenesis and ketogenesis. The immediate pharmacologic result is an increase in blood glucose at the expense of stored hepatic glycogen. The onset of action post injection is 5-20 minutes. Glucagon is degraded in the liver, kidney, and tissue receptor sites. The half life of glucagon in plasma is 3 to 6 minutes, similar to that of insulin.
Glucagon is soluble in aqueous solutions at pH less than 3 or greater than 9, and has low solubility in the pH range of 4 to 8 due to its isoelectric point of 7.1. It forms a gel in acidic aqueous conditions (pH 3-4) and precipitates within an hour of preparation in a neutral aqueous solution.
Currently, the commercial preparation of glucagon is a two part sterile vial, intended for immediate use following reconstitution. It is sold as a rescue kit and is available for intravenous, intramuscular or subcutaneous administration. The kit contains 1 mg (1 unit) of glucagon and 49 mg of lactose in a sterile vial. The diluent contains 12 mg/mL glycerin, water for injection and hydrochloric acid. The diluent is injected into the powder vial, gently swirled to dissolve the glucagon, then the glucagon solution is pulled back into the same syringe ready for injection. The pH of this solution is approximately 2. The recommended dose is typically 0.5-1 mg. Any reconstituted glucagon is to be discarded since it is not stable in solution.
Previous attempts to stabilize glucagon include the addition of cationic or anionic monovalent detergents to enhance the solubilization of 1 mg/mL glucagon using a 6 fold molar excess of detergent, as described in GB Patent No. 1202607; hen egg lysolecithin, which shows the detergent induced partial helical structure in solutions of glucagon containing about 0.02 mg/ml peptide, as described in J. Biol. Chem. 247, 4986-4991; 4992-4996 (1972); lysolecithin, as described in Biopolymers 21, 1217-1228 (1982), Biopolymers 22, 1003-1021 (1983); micelles of anionic detergent SDS at low pH, as described in Biochem. 19, 2117-2122 (1980), and at neutral pH, as described in Biochim. Biophys. Acta 603, 298-312 (1980); and cyclodextrins (J. Pharm Sci. 97(7):2720-9 (2008)); Eur J Pharm Sci. 2; 36 (4-5):412-20 (2009). EP 1061947 by Novo Nordisk describes stabilized glucagon solutions containing surfactant such as LPMC or other detergents carrying multiple charges (two or more negative, two or more positive, or both positive and negative) added in 0.5-20 moles detergent/peptide), solubilizing glucagon at pharmaceutically relevant concentrations in the entire pH range of 4 to 9. U.S. Pat. No. 5,652,216 to Kornfelt, et al., describes a pharmaceutical preparation comprising glucagon and a stabilizing amount of a pharmaceutically acceptable ampholyte such as an amino acid or dipeptide or a mixture thereof and optionally an excipient.
Recently, glucagon is being developed for use in an “artificial pancreas” or bihormonal pump. Insulin pumps have been used by insulin dependent diabetics for over a decade. These pumps are capable of providing a continuous flow of insulin to cater to their basal insulin needs. After eating, the user can manually increase the insulin flow to temporarily cover their meal, then cut back to the slow basal flow. These apparatus are attached to the abdominal surface by a small needle and may remain in place for up to a week. Newer devices also have been developed that combine the ability to read the patient glucose levels and deliver insulin as needed to cover individual patient requirements. However, should too much insulin be given, there is no way to prevent hypoglycemia. Therefore, the next step to complete the artificial pancreas is to add a second pump to deliver glucagon to the patient to counteract hypoglycemia. This creates an artificial pancreas capable of keeping a patient within ideal glucose levels, similar to how a normal functioning pancreas does in a non-diabetic individual. However, this application requires a glucagon that is stable in solution for at least seven days at 30-37° C., and the current commercial formulations are not capable of fulfilling that need. Moreover, since the currently available formulation is designed for “rescue” use, the acidic nature and pain of the injection is acceptable since it is a single dose, rarely given to the patient. However, the pH and isotonicity of the solution should be closer to physiological conditions for use in a pump.
It is therefore an object of the present invention to provide a glucagon that is stable as a clear solution for at least seven days at 37° C. for extended use in a pump device.